The present invention relates to a protein having an antithrombotic activity and a method for producing the same. The present invention also relates to DNA coding for the protein and a drug containing the protein as an active ingredient.
The number of patients with thromboses such as myocardial infarction and cerebral thrombosis, in particular, arterial thrombosis, is high in the world, and these are very important diseases to be treated. In an early stage of onset of arterial thrombosis, von Willebrand factor in blood binds to subendothelial tissues (collagen etc.) exposed due to impairment of vascular endothelial cells, and a membrane glycoprotein on platelets, glycoprotein Ib, binds to the von Willebrand factor. Thus, the platelets adhere to blood vessel walls and they are activated (J. P. Cean et al., J. Lab. Clin. Med., 87, pp. 586–596, 1976; K. J. Clemetson et al., Thromb. Haemost., 78, pp. 266–270, 1997). Therefore, it is an important target for antithrombotic drugs for treating or preventing thromboses to inhibit the binding of von Willebrand factor and glycoprotein Ib. However, there are few substances that have proven to exhibit antithrombotic property by inhibiting the binding of these proteins, and such drugs have not been used in clinical practice. It has been reported that a recombinant protein VCL that has a sequence of the 504th to 728th amino acid residues of the amino acid sequence of von Willebrand factor shows an antithrombotic action by inhibiting the binding of von Willebrand factor and glycoprotein Ib (K. Azzam et al., Thromb. Haemost., 73, pp. 318–323, 1995). Further, it has also been reported that a monoclonal antibody AJvW-2 directed to human von Willebrand factor exhibits an antithrombotic activity by specifically binding to von Willebrand factor without showing hemorrhagic tendency (S. Kageyama et al., Br. J. Pharmacol., 122, pp. 165–171, 1997). It has also been shown that a monoclonal antibody 6B4 directed to glycoprotein Ib has an antithrombotic action in animal models (N. Cauwenberghs et al., Atherioscler. Thromb. Vasc. Biol., 20, pp. 1347–1353, 2000). Furthermore, the protein AS1051 originating from snake venom specifically binds to platelet glycoprotein Ib to similarly exhibit antithrombotic property without showing hemorrhagic tendency (N. Fukuchi et al., WO95/08573).
Meanwhile, the binding of von Willebrand factor and glycoprotein Ib is not observed under a usual condition, but is considered to occur only under a condition where shear stress is induced, such as a condition in a blood flow (T. T. Vincent et al., Blood, 65, pp. 823–831, 1985). However, as a method for artificially observing the binding of these proteins, there are known addition of an antibiotic, ristocetin (M. A. Howard and B. G. Firkin, Thromb. Haemost., 26, pp. 362–369, 1971) and addition of a protein originating from snake venom, botrocetin (M. S. Read et al., Proc. Natl. Acad. Sci. USA., 75, pp. 4514–4518, 1978). That is, both of the substances are considered to cause a structural change of von Willebrand factor by binding to a specific site of von Willebrand factor, thereby causing the binding of von Willebrand factor and glycoprotein Ib, which does not occur under a usual condition.
As proteins originating from snake venom, there are known, in addition to the aforementioned AS1051 (derivative of the α-chain of a protein originating from Crotalus horridus horridus snake venom, CHH-B) and its original protein, CHH-B, many glycoprotein Ib-binding proteins such as alboaggregin, echicetin, mamushigin, jararaca-GPIbBP and proteins originating from Cerastes cerastes. Many of these proteins have a heterodimeric structure, and the amino acid sequences of their subunits show a homology of not less than 30%. Furthermore, they are proteins in which all of subunits show an amino acid sequence homology of not less than 30% to the CHH-B α-chain (R. K. Andrews et al., Biochemistry, 35, pp. 12629–12639, 1996; Y. Fujimura et al., Thromb. Haemost., 76, pp. 633–639, 1996).
While such glycoprotein Ib-binding proteins originating from snake venom that inhibit the binding of glycoprotein Ib and von Willebrand factor and monoclonal antibodies directed to von Willebrand factor or glycoprotein Ib are known to exhibit an antithrombotic action as described above, some of proteins originating from snake venom that bind to a platelet membrane glycoprotein, glycoprotein IIb/IIIa, disintegrins (T. Matsui et al., Biochem. Biophys. Acta, 1477, pp. 146–56, 2000) and monoclonal antibodies directed to glycoprotein IIb/IIIa (A. M. Lincoff et al., J. Am. Coll. Cardiol., 35, pp. 1103–1115, 2000) have also been shown to exhibit an antithrombotic activity in animal experiments or clinical practice. For example, a peptide prepared from a disintegrin sequence, Eptifibatide (integrilin), has been shown to have clinical efficacy as an antithrombotic drug. Further, a chimerized monoclonal antibody directed to glycoprotein IIb/IIIa, Abciximab (ReoPro), is also widely used as an antithrombotic drug in clinical practice and its strong antithrombotic action and its therapeutic action for acute coronary syndromes have been reported (M. Madan et al., Circulation, 98, pp. 2629–2635, 1998).
In addition to the above proteinaceous substances, low molecular weight organic compounds that bind to a platelet membrane glycoproteins and inhibit their function are known with respect to glycoprotein Ib (N. Fukuchi et al., WO99/54360; W. Mederski et al., WO00/32577; H. Matsuno et al., Circulation, 96, pp. 1299–1304, 1997) and glycoprotein IIb/IIIa (E.J. Topol et al., Lancet, 353, pp. 227–231, 1999). Among these, some of glycoprotein IIb/IIIa antagonists are clinically used, but they have not been shown to have efficacy as high as that of Abciximab (ReoPro) (E. J. Topol et al., Lancet, 353, pp. 227–231, 1999; M. Madan et al., Circulation, 98, pp. 2629–2635, 1998).
As described above, proteins that bind to platelet membrane proteins involved in thrombogenesis such as glycoprotein Ib and glycoprotein IIb/IIIa and inhibit their functions in thrombogenesis are useful as antithrombotic drugs, and many exogenous proteins have been developed as antithrombotic drugs. Among these, a chimerized monoclonal antibody directed to glycoprotein IIb/IIIa, Abciximab (ReoPro), shows high clinical efficacy. However, the following some conditions are still required to use proteinaceous substances, in particular, exogenous proteins as clinically usable drugs.
(1) High Binding Activity to Target
In the case of Abciximab (Reopro), a high binding activity to platelets (glycoprotein IIb/IIIa) can be mentioned as one of the reasons for its high efficacy (R. M. Scarborough et al., Circulation, 100, pp. 437–444, 1999). That is, it is considered that the administered Abciximab (ReoPro) firmly binds to platelets and as a result, it exists in blood together with platelets for a long period, thereby showing drug efficacy for a long period.
(2) Long Half-Life/High Drug Efficacy Retention in Blood
For administration of a proteinaceous drug, in particular, a drug that is not originally an endogeneous substance existing in the organisms, repetitive administration is generally difficult and a single administration is usually performed. Therefore, drug efficacy must be maintained for a certain long period and long half-life and/or high drug efficacy retention in blood is required.
(3) Low Antigenicity
Even when a single administration is performed, low antigenicity is required so that an excessive antigen-antibody reaction should not occur.
(4) Useful Actions in Addition to Main Action
There have been reported that Abciximab (ReoPro) actually has a binding action directed to other proteins such as αvβ3 integrin and Mac-1 in addition to an inhibitory action directed to glycoprotein IIb/IIIa (B. S. Coller, Thromb. Haemost., 82, pp. 326–336, 1999). It is considered that this secondary action is one of the reasons for high clinical efficacy. That is, clinical efficacy of a drug may be increased by acting on several targets other than a single target.
It has been reported that a drug for inhibiting the binding of glycoprotein Ib and von Willebrand factor has a low risk of hemorrhage compared with a drug for inhibiting the function of glycoprotein IIb/IIIa (S. Kageyama et al., Br. J. Pharmacol., 122, pp. 165–171, 1997), and therefore it can be a useful antithrombotic drug. Among the aforementioned proteins that inhibit the binding of glycoprotein Ib and von Willebrand factor, monoclonal antibodies generally have a high binding activity (affinity) to a target and can satisfy the above requirements (2) and (3) if they are modified into a chimera antibody or a humanized antibody. On the other hand, it is considered that proteins other than the monoclonal antibodies, for example, a glycoprotein Ib-binding protein originating from snake venom have a low binding activity (affinity) to their targets. For example, when the anti-platelet activity disclosed for a protein derivative originating from snake venom, AS1051 (N. Fukuchi et al., WO95/08573), is compared with that of a monoclonal antibody, AJvW-2 (S. Kageyama et al., Br. J. Pharmacol., 122, pp. 165–171, 1997), the binding activity (affinity) of AS1051 on a molar concentration basis is calculated to be about 1/10 based on the fact that the efficacy is shown at almost the same concentration (weight concentration), and the molecular weight of AS1051 is about 15,000 Da and that of the monoclonal antibody about 150,000 Da.
Further, the present inventors found that, as shown in the examples described later, repetitive administration of AS1051 produces antibodies for AS1051 as an antigen and subsequent administration thereof caused platelet decrease that was considered to be attributable to the antibody generation.
That is, in order to clinically use glycoprotein Ib-binding proteins originating from snake venom such as AS1051 as antithrombotic drugs, they must further be improved for the aforementioned requirements (1) to (3).